Differential N- and O-glycosylation signatures of HIV-1 Gag virus-like particles and coproduced extracellular vesicles

Abstract

Human immunodeficiency virus 1 (HIV-1) virus-like particles (VLPs) are nanostructures derived from the self-assembly and cell budding of Gag polyprotein. Mimicking the native structure of the virus and being noninfectious, they represent promising candidates for the development of new vaccines as they elicit a strong immune response. In addition to this, the bounding membrane can be functionalized with exogenous antigens to target different diseases. Protein glycosylation depends strictly on the production platform and expression system used and the displayed glycosylation patterns may influence downstream processing as well as the immune response. One of the main challenges for the development of Gag VLP production bioprocess is the separation of VLPs and coproduced extracellular vesicles (EVs). In this study, porous graphitized carbon separation method coupled with mass spectrometry was used to characterize the N- and O- glycosylation profiles of Gag VLPs produced in HEK293 cells. We identified differential glycan signatures between VLPs and EVs that could pave the way for further separation and purification strategies to optimize downstream processing and move forward in VLP-based vaccine production technology.

Keywords: HIV-1; N-glycans; O-glycans; porous graphitized carbon; vaccine; virus-like particles.

Figure 1

Experimental design and virus‐like particle (VLP) production analysis. (a) Experimental workflow. Two biological replicates of HEK293 cell cultures were grown under three different conditions: no transfected condition, transfected with an empty plasmid denoted as “mock,” and transfected using the standard protocol with the plasmid coding for the translational fusion of HIV‐1 Gag polyprotein and enhanced GFP (Gag::eGFP) polyprotein. Both transfections were performed at the same cell density of 2 × 10⁶ cells/ml. At the time point of 72 h post‐transfection (hpt), cultured samples were taken and centrifuged and supernatants were ultracentrifuged at 31,000 rpm using a 30% sucrose cushion. The resulting pellet was resuspended in 1 ml of phosphate‐buffered saline (PBS). The concentrated sample was subjected to N‐ and O‐glycan release and reduction. Resulted alditols were analyzed by porous grafitized carbon liquid chromatography with electrospray ionization tandem mass spectrometry (PGC‐LC‐ESI‐MS/MS) to assign the glycosylation structures. (b) Concentration via ultracentrifugation measured in relative fluorescence units (RFUs). Harvested VLPs were concentrated 12.2‐fold in the analytical ultracentrifugation step. (c) Extracted ion chromatograms profiles of samples that underwent lysis treatment and samples that contained intact VLPs for glycan analysis (no lysis treatment)

Figure 2

Analysis of N‐glycan species from Gag virus‐like particles (VLPs). (a) Combined extracted ion chromatograms of 51 N‐glycan species released from VLP samples. Structures of the 17 most abundant glycans are depicted. Complex glycans are represented in blue, hybrid glycans represented in red, and oligomannoses or high mannose glycans represented in green. (b) Classification of the different types of N‐glycan species identified in the sample. Glycans with sulfate and Lewis epitopes represent a subdivision within complex glycans. (c) Relative abundance distribution of the 12 most abundant N‐glycan species identified in the VLP sample. Error bars represent standard deviation. The average value of all 51 glycans (1.96) is represented by the dotted line. F, fucose; H, hexose; N, N‐acetylhexosamine; S, N‐acetylneuraminic acid, Su: sulfate

Figure 3

Analysis of O‐glycan species from Gag virus‐like particles (VLPs). (a) Combined extracted ion chromatograms of nine O‐glycan species released from VLP samples in which the top five most abundant glycans account for 94% of relative abundance. (b) Classification of the different types of O‐glycan species identified in the sample. (c) Relative abundance of the nine O‐glycan species identified in the VLP sample. Error bars represent standard deviation. Significance calculated by two‐way analysis of variance. H, hexose; N, N‐acetylhexosamine; S, N‐acetylneuraminic acid; Su, sulfate

Figure 4

Example of the porous graphitized carbon‐liquid chromatography separation of O‐glycan structural isomers and MS2 analysis. (a) Extracted ion chromatogram (EIC) of the m/z 1040.52^{1 −} (Hex₂ HexNAc₂ NeuAc₁) O‐glycan isomers. (b) MS2 spectrum of the m/z 1040.52^{1 −} isomer observed at RT 58.1 min. (c) MS2 spectrum of the m/z 1040.52^{1 −} isomer observed at RT 66.1 min. Diagnostic ions are denoted in red. H, hexose; N, N‐acetylhexosamine; S, N‐acetylneuraminic acid

Figure 5

Relative abundance changes in glycans of produced particles upon transfection and virus‐like particle production. (a) Heatmap illustrating relative abundance and fold change ratios of N‐glycans showing changes in the different studied conditions (left). Replicates are denoted as I, II, III, and IV. (b) Heatmap illustrating relative abundance and fold change ratios of O‐glycans showing changes in the different studied conditions (left). Replicates are denoted as I, II, III, and IV. F, fucose; H, hexose; N, N‐acetylhexosamine; S, N‐acetylneuraminic acid; Su, sulfate

Figure 6

Example of the porous graphitized carbon‐liquid chromatography separation of N‐glycan structural isomers and MS2 analysis. (a) Extracted ion chromatogram (EIC) of the m/z 832.81^{2 −} (Hex₃ HexNAc₅ Fuc₁) N‐glycan isomers. (b) MS2 spectrum of the m/z 832.81^{2 −} isomer observed at RT 49.8 min. (c) MS2 spectrum of the m/z 832.81^{2 −} isomer observed at RT 59.9 min. Diagnostic ions are denoted in red. F, fucose; GlcNAc, N‐Acetylglucosamine; H, hexose; N, N‐acetylhexosamine

Figure 7

Analysis of diffracting particles. (a) The size distribution curve of diffracting particles in each condition measured by nanoparticle tracking analysis (NTA). The vertical gray line represents 145 nm. N: nontransfected condition, M: condition transfected with mock plasmid, S: condition transfected with plasmid encoding translational fusion of HIV‐1 Gag polyprotein and enhanced GFP (Gag::eGFP) polyprotein, VLPs: virus‐like particles, EVs: extracellular vesicles. (b) Glycan density per particle. The total intensity area of identified glycans in each condition was normalized by their corresponding internal standard DP7 intensity area and divided by the corresponding number of particles in the sample. The number of particles was calculated using the particle concentration value obtained by NTA. Significance was calculated using two‐way analysis of variance. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; ns: non significant. (c) Schematic representation of the glycan density in EVs (left) and Gag VLPs (right). The mean value of the particle diameter measured by NTA is represented within the particle. The represented glycan species in EVs (left) are the most abundant and the ones showing downregulation in S condition. The represented glycans in VLPs (right) are the ones showing an increase in S condition. The values showing the number glycan/particle are the approximate value calculated assuming the same response factor of the standard DP7 with known concentration, used in each condition